Differential effects of SNAP-25 deletion on Ca2+-dependent and Ca2+-independent neurotransmission

Department of Neuroscience, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111, USA.
Journal of Neurophysiology (Impact Factor: 3.04). 09/2007; 98(2):794-806. DOI: 10.1152/jn.00226.2007
Source: PubMed

ABSTRACT At the synapse, SNAP-25, along with syntaxin/HPC-1 and synaptobrevin/VAMP, forms SNARE N-ethylmaleimide-sensitive factor [soluble (NSF) attachment protein receptor] complexes that are thought to catalyze membrane fusion. Results from neuronal cultures of synaptobrevin-2 knockout (KO) mice showed that loss of synaptobrevin has a more severe effect on calcium-evoked release than on spontaneous release or on release evoked by hypertonicity. In this study, we recorded neurotransmitter release from neuronal cultures of SNAP-25 KO mice to determine whether they share this property. In neurons lacking SNAP-25, as those deficient in synaptobrevin-2, we found that approximately 10-12% of calcium-independent excitatory and inhibitory neurotransmitter release persisted. However, in contrast to synaptobrevin-2 knockouts, this remaining readily releasable pool in SNAP-25-deficient synapses was virtually insensitive to calcium-dependent-evoked stimulation. Although field stimulation reliably evoked neurotransmitter release in synaptobrevin-2 KO neurons, responses were rare in neurons lacking SNAP-25, and unlike synaptobrevin-2-deficient synapses, SNAP-25-deficient synapses did not exhibit facilitation of release during high-frequency stimulation. This severe loss of evoked exocytosis was matched by a reduction, but not a complete loss, of endocytosis during evoked stimulation. Moreover, synaptic vesicle turnover probed by FM-dye uptake and release during hypertonic stimulation was relatively unaffected by the absence of SNAP-25. This last difference indicates that in contrast to synaptobrevin, SNAP-25 does not directly function in endocytosis. Together, these results suggest that SNAP-25 has a more significant role in calcium-secretion coupling than synaptobrevin-2.

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Available from: Michael C Wilson, Aug 16, 2015
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    • "However, even a slight increase in VAMP proteins levels could accommodate successful vesicle fusion (van den Bogaart et al., 2010). Loss of function studies of key SNAREs involved in fusion in neuronal system suggest that a parallel pathway, possibly involving noncanonical SNAREs typically implicated in constitutive vesicle trafficking, may mediate fusion and recycling of a subset of vesicles (Bronk et al., 2007; Deak et al., 2004; Schoch et al., 2001; Washbourne et al., 2002; Ramirez and Kavalali, 2012). Certainly, there was no clear transcriptional activation of any of the SNARE genes that we examined in the knockdowns . "
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    ABSTRACT: Exocytosis involves membrane fusion between secretory vesicles and the plasma membrane. The Soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs) and their receptor proteins (SNAREs) interact to fuse vesicles with the membrane and trigger the release of their sialosecretome out of the tick salivary gland cells. In this study, we examined the functional significance of the Vti family of SNARE proteins of blood-feeding Amblyomma maculatum and A. americanum. Vti1A and Vti1B have been implicated in multiple functional roles in vesicle transport. QRT-PCR studies demonstrated that the highest transcriptional expression of vti1a and vti1b genes occurs in unfed salivary glands, suggesting that elevated secretory vesicle formation occurs prior to feeding but continues at low rates after blood feeding commences. Vti1A and Vti1B localize to the secretory vesicles in unfed tick salivary glands in immunofluorescence microscopy studies. Knockdown of vti1a and vti1b by RNA interference resulted in a significant decrease in the engorged tick weight compared to the control during prolonged blood-feeding on the host. RNA interference of vti1a or vti1b impaired oviposition and none of the ticks produced eggs masses. Surprisingly, the double knockdown did not produce a strong phenotype and ticks fed normally on the host and produced egg masses, suggesting a compensatory mechanism exists within the secretory system which may have been activated in the double knockdown. These results suggest an important functional role of the Vti family of SNARE proteins in tick blood feeding and ultimately oviposition. Understanding the basic functions of the Vti family of SNARE proteins in salivary glands may lead to better ways to prevent tick attachment and transmission of tick-borne diseases.
    Insect biochemistry and molecular biology 03/2013; 43(5). DOI:10.1016/j.ibmb.2013.03.003 · 3.42 Impact Factor
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    • "For example, loss of SNAP25 or synaptobrevin largely abolishes calcium-dependent evoked release but leaves spontaneous fusion release intact suggesting a role for alternate SNAREs in mediating spontaneous release (Schoch et al., 2001; Washbourne et al., 2002; Bronk et al., 2007). The CLASP2-mediated increase in synapsin synaptobrevin, and SNAP25 may provide possible mechanistic detail of the functional increase in evoked mEPSCs as alterations in expression levels of these proteins have been shown to cause calcium-dependent evoked neurotransmitter release (Lu et al., 1992; Rosahl et al., 1995; Schoch et al., 2001; Washbourne et al., 2002; Bronk et al., 2007). "
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    ABSTRACT: Microtubule organization and dynamics are essential during axon and dendrite formation and maintenance in neurons. However, little is known about the regulation of microtubule dynamics during synaptic development and function in mammalian neurons. Here, we present evidence that the microtubule plus-end tracking protein CLASP2 (cytoplasmic linker associated protein 2) is a key regulator of axon and dendrite outgrowth that leads to functional alterations in synaptic activity and formation. We found that CLASP2 protein levels steadily increase throughout neuronal development in the mouse brain and are specifically enriched at the growth cones of extending neurites. The short-hairpin RNA-mediated knockdown of CLASP2 in primary mouse neurons decreased axon and dendritic length, whereas overexpression of human CLASP2 caused the formation of multiple axons, enhanced dendritic branching, and Golgi condensation, implicating CLASP2 in neuronal morphogenesis. In addition, the CLASP2-induced morphological changes led to significant functional alterations in synaptic transmission. CLASP2 overexpression produced a large increase in spontaneous miniature event frequency that was specific to excitatory neurotransmitter release. The changes in presynaptic activity produced by CLASP2 overexpression were accompanied by increases in presynaptic terminal circumference, total synapse number, and a selective increase in presynaptic proteins that are involved in neurotransmitter release. Also, we found a smaller increase in miniature event amplitude that was accompanied by an increase in postsynaptic surface expression of GluA1 receptor localization. Together, these results provide evidence for involvement of the microtubule plus-end tracking protein CLASP2 in cytoskeleton-related mechanisms underlying neuronal polarity and interplay between microtubule stabilization and synapse formation and activity.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2012; 32(40):13906-16. DOI:10.1523/JNEUROSCI.2108-12.2012 · 6.75 Impact Factor
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    • "or in inhibitory (mIPSCs) and excitatory (mEPSCs) spontaneous release events (Supplementary Figure S2D–I). Thus, a loss of the vast majority of SNAP-25 from neurons does not cause a major synaptic phenotype, as opposed to a complete loss of SNAP-25 in KO mice that blocks synaptic transmission (Washbourne et al, 2002; Bronk et al, 2007). This surprising result suggests that the small amount of reserve SNAP-25 protein remaining in KD neurons is largely sufficient for full presynaptic function. "
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    ABSTRACT: At a synapse, the synaptic vesicle protein cysteine-string protein-α (CSPα) functions as a co-chaperone for the SNARE protein SNAP-25. Knockout (KO) of CSPα causes fulminant neurodegeneration that is rescued by α-synuclein overexpression. The CSPα KO decreases SNAP-25 levels and impairs SNARE-complex assembly; only the latter but not the former is reversed by α-synuclein. Thus, the question arises whether the CSPα KO phenotype is due to decreased SNAP-25 function that then causes neurodegeneration, or due to the dysfunction of multiple as-yet uncharacterized CSPα targets. Here, we demonstrate that decreasing SNAP-25 levels in CSPα KO mice by either KO or knockdown of SNAP-25 aggravated their phenotype. Conversely, increasing SNAP-25 levels by overexpression rescued their phenotype. Inactive SNAP-25 mutants were unable to rescue, showing that the rescue was specific. Under all conditions, the neurodegenerative phenotype precisely correlated with SNARE-complex assembly, indicating that impaired SNARE-complex assembly due to decreased SNAP-25 levels is the ultimate correlate of neurodegeneration. Our findings suggest that the neurodegeneration in CSPα KO mice is primarily produced by defective SNAP-25 function, which causes neurodegeneration by impairing SNARE-complex assembly.
    The EMBO Journal 12/2011; 31(4):829-41. DOI:10.1038/emboj.2011.467 · 10.75 Impact Factor
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